Characterizing Pathology in Erythrocytes

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Characterizing Pathology in Erythrocytes Characterizing pathology in erythrocytes using morphological and biophysical membrane properties: relation to impaired hemorheology and cardiovascular function in rheumatoid arthritis Oore-ofe O Olumuyiwa-Akeredolu1, Prashilla Soma2, Antoinette V Buys3, Legesse Kassa Debusho4, Etheresia Pretorius5,* 1Department of Physiology, Faculty of Health Sciences, University of Pretoria, Private Bag X323, Arcadia, 0007, RSA. [email protected] 2Department of Physiology, Faculty of Health Sciences, University of Pretoria, Private Bag X323, Arcadia, 0007, RSA. [email protected] 3Unit for Microscopy and Microanalysis, Department of Anatomy and Physiology, Faculty of Veterinary Sciences, University of Pretoria. [email protected] 4Department of Statistics, University of South Africa, Pretoria RSA. [email protected] 5Department of Physiological Sciences, Stellenbosch University, Private Bag X1, Matieland, 7602, RSA. [email protected] *Corresponding Author: Etheresia Pretorius Department of Physiological Sciences Faculty of Natural Sciences Stellenbosch University Stellenbosch South Africa Phone: 27 829295041 Fax: 27 21 808 3145 E-mail: [email protected] Highlights • Erythrocytes (RBCs) in rheumatoid arthritis (RA) have reduced elasticity. • RBCs in RA display a wide range of poikilocytosis. • RBCs in RA display membrane pathomorphology. • Band 3 skeletal protein network is altered in RA RBCs. • Specific poikilocytes have been identified frequently with the use of NSAIDs or the presence of cardiovascular comorbidities. Authorship/Acknowledgments OOA performed the research study, analysed and interpreted its outcomes and wrote this paper. PS was the physician who screened, selected, and drew blood from all study participants. LKD was the statistician who performed analyses for data obtained from AFM studies. EP designed and appraised the study and its outcomes. AVB performed collation of the AFM data. Assistance with utility of microscopes was provided by staff of the Laboratory for Microscopy and Microanalysis, University of Pretoria. All authors read and approved the manuscript. 1 | P a g e Funding Medical Research Council: E Pretorius (fund number A0X331) and National Research Foundation: E Pretorius (fund number: N00345). Graphical abstract Abstract The inflammatory burden of the complex Rheumatoid Arthritis (RA) disease affects several organ-systems, including rheological properties of blood and its formed elements. Red blood cells (RBCs) are constantly exposed to circulating dysregulated inflammatory molecules that are co-transported within the vasculature; and their membranes may be particularly vulnerable to the accompanying oxidative stress. In the current study, we investigate biophysical and ultrastructural characteristics of RBCs obtained from a cohort of patients using atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy (CM). Statistical analyses of AFM data showed that RA RBCs possessed significantly reduced membrane elasticity relative to that of RBCs from healthy individuals (P-value < 0.0001). SEM imaging of RA RBCs revealed increased anisocytes and poikilocytes. Poikilocytes included knizocytes, stomatocytes, dacryocytes, irregularly contracted cells, and knot cells. CM imaging of several RA RBCs, spectrin, and band 3 protein 2 | P a g e networks portrayed the similar morphological profiles. Analyses of CM images confirmed changes to distribution of band-3 skeletal protein, a protein critical for gaseous exchange functions of the RBC and preventing membrane surface loss. Decreased membrane deformability impairs the RBC’s capacity to adequately adapt its shape to navigate blood vessels, especially microvasculature, and this decrease is also reflected in the cell’s morphology. Changes to morphology and deformability may also indicate loss of functional domains and/or pathological protein and lipid associations. These findings suggest that RA disease and/or its concomitant factors impact on the RBC and its membrane integrity with potential for exacerbating pathological cellular function, hemorheology, and cardiovascular function. Keywords Rheumatoid arthritis, erythrocyte, membrane, poikilocytes, atomic force microscopy, rheology. 1. INTRODUCTION Rheumatoid arthritis (RA) is an inflammatory autoimmune disease with complex aetiology and pathobiology and varied manifestations across affected individuals although a common pathological feature is changed blood rheology, frequently manifesting as raised RBC sedimentation rates. The inflammatory burden of RA affects several organ-systems in addition to its articular influences. The circulatory system and its cells are suspected to be less-detected extraarticular targets of the disease and its inflammatory mediators, such as IL-6, which has been demonstrated to change the morphological features of healthy blood (1, 2). There is ample evidence that RA-induced inflammation inflicts oxidative assault on the RBC and affects its biochemical and physiological activities within circulation (3-7). Free radicals in blood reduce protective membrane thiols and antioxidant enzymes like 3 | P a g e superoxide dismutase while generating dysfunction in the ion transport ATPases of RA RBCs (8). Rheology and hemodynamics of blood are compounded by morphopathological changes to the RBC membrane (9). Capacity for gaseous exchange functions of integral proteins and overall skeletal integrity may also be compromised by membrane damage (10, 11). RBC-associated indices correlate with some markers of inflammation in RA. Among those reported are RBC sedimentation rate (ESR), haemoglobin levels, and RBC counts, which were found to correlate with levels of radiographic erosion (12, 13). Elevated fibrinogen concentration and IgG levels in RA patients have been shown to correlate with increased plasma viscosity (14). Increased ESR and fibrinogen levels are also hemorheologic markers and indicators of RA disease progression (15). These markers implicate the autonomous contribution of RBCs morphopathology, besides other factors in plasma, to hemorheological disorders in RA. Of late, the Center for Disease Control and Prevention reported an ongoing rise in arthritis prevalence in the United States adult population, amongst which RA is included (16). Cardiovascular (CV) diseases have been demonstrated to be a significant source of mortality in affected persons. In 2016, the European League Against Rheumatism taskforce made recommendations for expert opinion-based screening for CV risk factors (17). Besides vascular and cardiac tissue integrity, blood and especially the RBC, is critical to performing this critical assignment of CV risk assessment considering it makes up over 90% of formed elements within blood. In cardiovascular diseases, RBC indices have been implicated in impaired blood rheology. A direct relationship has been found between decreased RBC deformability and increased risk for arterial hypertension in non-RA conditions (18). RBC distribution width (RDW) values above a 14% threshold were associated with 4 | P a g e decreased cellular deformability (19). A parallel association was also found between RDW and the occurrence of acute myocardial infarction in RA patients excluding individuals with other cardiovascular diseases (20). Under conditions of increased shear stress, as typically occur in microvasculature, RBCs’ deformability determine efficacy of tissue perfusion, but even shape and surface attributes determine optimized blood flow rate in larger vessels (9). As discussed, the RBC’s biophysical properties are critical to blood rheology, RA pathophysiology, and the occurrence of cardiovascular comorbidities. Subsequently, we discuss its structure and function. 1.1 Erythrocyte Membrane Structure The RBC membrane comprises a phospholipid bilayer, an underlying supporting skeleton, integral, transmembrane, and anchoring biomolecules. Two key components of underlying skeleton, which are critical to and indicative of the integrity of the cell’s structure, are the band 3 integral membrane protein and the cytoskeletal spectrin network. Anion exchanger 1 or band 3 is an important biomolecular switch that mediates oxygen-regulation of the cell’s functions such as the assembly of crucial metabolic enzymes, interaction of ankyrin with band 3, and ATP release (21). A hexagonal spectrin lattice comprising interwoven α and β chains is anchored by ankyrin to a complex made up of band 3, CD47, protein 4.2 (pallidin) and Rh proteins, which all contribute to the cell’s structural integrity (10, 22, 23). Other membranous biomolecular inclusions are flippase, floppase, and scramblase which control the transfer of phospholipids from inner to outer leaflets, lipid rafts comprising stomatin, tightly packed outer leaflet sphingolipids, the glycosylphosphatidylinositol- bound enzyme acetylcholinesterase, inner leaflet phosphatidylserine and 5 | P a g e phosphatidylethanolamine with which flotillin non-covalently associates while cholesterol interacts with hydrophobic tails (24). Cholesterol is embedded between the membrane leaflets, and its augmentation within this space results in decreased cellular permeability and fluidity (25, 26). There are also inorganic molecule transporters such as: glut 1 anchored to the actin-tropomyosin-tropomodulin complex by dematin and adducin, glycophorin anchored by protein p55 to protein 4.1 and the spectrin network, calcium pumps, and aquaporins (24). A pathological coagulation system, including damage to RBCs, are hallmarks of
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